Liposomes are small vesicles comprising amphipathic lipids arranged in spherical bilayers. Liposomes may contain many concentric lipid bilayers separated by aqueous channels (multilamellar vesicles or MLVs), or alternatively, they may contain a single membrane bilayer (unilamellar vesicles), which may be small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs). The lipid bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. In the membrane bilayer, the hydrophobic "tails" of the lipid monolayers orient towards the center of the bilayer, whereas the hydrophilic "heads" orient toward the aqueous phase.
Liposomes may be used to encapsulate a variety of materials by trapping hydrophilic compounds in the aqueous interior or between bilayers, or by trapping hydrophobic compounds within the bilayer. As such, they are particularly useful to deliver biologically active materials by encapsulating compounds which exhibit poor aqueous solubility or which exhibit unacceptable toxicity at therapeutic dosages.
Liposomes and related phospholipid vesicle complexes can be prepared by a variety of techniques. In general, these techniques start with "dry" lipids that are introduced into an aqueous phase (D. Lasic, J.Theor. Biol. (1087) 124:35-41). Once the lipid is hydrated, liposomes form spontaneously. Techniques have been developed to control the number of lamellae in the liposome and to produce defined particle size. U.S. Pat. No. 4,935,171 discloses a single-vessel method of preparing liposomes by forming a homogeneous lipid-film in a thin-film evaporator by evaporation of the organic solvent, followed by in-situ hydration of the film in an aqueous phase by agitation.
Another method of preparing liposomes is known as the ethanolic injection process. (S. Batzre et al., Biochem. Biophys Acta (1973) 298:1015-1019; J. Kremer at al., Biochemistry (1977) 16:3932-3935). In this process, the lipids are dissolved in ethanol and injected into the aqueous phase, optionally containing a buffer solution. This process generates unilamellar or multilamellar vesicles of defined size. One advantage of this process is that it permits continuous production of vesicles if the solvent can be removed in a continuous process. Generally, the procedure requires removal of the solvent following injection of the solvent into the aqueous phase. This is generally accomplished by dialysis or other filtration processes such as tangential flow filtration.
Pulmonary surfactant is a complex mixture of lipids and proteins that promotes the formation of a monolayer at the alveolar air-water interface and, by reducing the surface tension, prevents the collapse of the alveolus during expiration. Premature infants, and occasionally full term neonates, sometimes suffer from a condition known as respiratory distress syndrome (RDS) due to the lack of sufficient endogenous pulmonary surfactant. Artificial pulmonary surfactants have therefore been developed to treat this condition thereby reducing infant morbidity and mortality.
One of these artificial pulmonary surfactants, known as KL4, is disclosed in U.S. Pat. Nos. 5,164,369 and 5,260,273, hereby incorporated by reference into the present application. Disclosed therein is a synthetic pulmonary surfactant composition comprising a pharmaceutically acceptable phospholipid admixed with a polypeptide having alternating hydrophobic and positively charged amino acid residues. As formulated for clinical use, the composition is a liposome comprised of dipalmitoyl-phosphatidylcholine (DPPC), palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA) and the synthetic peptide KL4 suspended in a buffered aqueous medium. The final drug product is a viscous suspension intended for direct instillation into the lung.
At present, this liposomal KL4 composition is formulated by the aforementioned ethanolic injection process. As described above, it is general practice to remove the ethanol solvent from the composition by dialysis. However, the dialysis procedure is not satisfactory for the liposomal KL4 drug product due to the viscous nature of the product. There is thus a need for a method of removing the ethanol from liposomal formulations prepared by ethanolic injection where the liposomal formulation is viscous as in the case of KL4. Advantageously, the solvent removal process should allow for continuous removal of the solvent during the manufacturing process.
In addition, it is noted that the extent of KL4 peptide insertion and the subsequent manner with which it associates with the components of the liposome lipid bilayer must be optimal for drug product performance with respect to both surface tension lowering properties and viscosity (fluidity). One of the problems encountered in the formulation and performance of liposomal KL4 drug product is that the viscosity of the drug product can limit effective distribution in the lung, thereby reducing in vivo activity. The present invention is intended to improve performance of KL4 liposomal pulmonary surfactant by facilitating protein insertion into the lipid bilayer and reducing viscosity of the final drug product.